The broad, long-term objective of the proposed project is to understand the basis of protein folding and stability. Beta sheets are a major structural component in proteins, but our understanding of the mechanism of beta-sheet formation is limited. Beta-sheet structures plays critical roles in amyloid fibrils associated with serious diseases and in protein aggregation. The focus of the project is on the stability and folding of a novel, single-layer beta-sheet found in outer surface protein A (OspA) from Borrelia burgdorfei. OspA is a predominantly beta-sheet protein, and it contains, in the middle of the molecule, a three-stranded beta- region that is solvent-exposed on both faces. We showed that this single layer beta-sheet is stably formed in solution, and that the single-layer beta-sheet can be extended by inserting a beta-hairpin unit. Thus, our system based on OspA provides a unique opportunity to study the molecular mechanism of beta-sheet formation in the absence of extensive long-range interactions. Our specific aims are: 1) To characterize the equilibrium and kinetic folding mechanisms of OspA. 2) To identify factors contributing to the stability of the single-layer beta sheet. 3) To identify new amino acid sequences that stably fold into the single-layer beta-sheet conformation. 4) To characterize the structures of OspA fragments that contain the N-terminal globular domain and single-layer beta-sheets of various length. 5) To characterize the equilibrium and kinetic folding mechanisms of these N-terminal fragments. 6) To study amyloid-like fibril formation of synthetic peptides derived from the single-layer beta-sheet. To achieve these goals, we will apply biophysical techniques including NMR spectroscopy, small angle X-ray scattering and differential scanning calorimetry, protein engineering and combinatorial library screening. These studies will provide new insights into the stability and folding of beta-structure, and into the mechanisms of amyloid fibril formation. Outcomes of this study will also provide important information for structural genomics and de novo protein design.